Tuberculosis (TB) is rivaled only by AIDS as a communicable cause of death. Infection is lifelong and may reactivate following an asymptomatic (latent) interval of variable duration. It is estimated that nearly two billion individuals worldwide have been exposed to Mycobacterium tuberculosis. Exposed (tuberculin-positive) individuals have a 5-10% lifetime risk of developing active TB. The risk rises to nearly 10% per year for individuals with HIV/AIDS, indicating a key role for the host immune response in maintaining TB latency. Chemoprophylaxis of latent TB to prevent reactivation requires months of drug therapy, a regimen that many individuals are unwilling or unable to complete. The development of better tools for TB control will hinge on the elucidation of the adaptive mechanisms that allow latent M. tuberculosis to persist in the face of host immunity and chemotherapy. Towards that goal, this application is focused on the identification of mycobacterial "persistence factors" and "defense factors" via signature-tagged mutagenesis (STM), a method for simultaneous screening of multiple mutants in mice. Two genetic screens are described. The persistence (per) screen will identify M. tuberculosis mutants that are specifically impaired in their ability to persist at later stages of infection in mice. The defense (def) screen will identify mutants whose attenuated phenotype in immune-competent mice is reversed in mice with specific immune deficiencies. These screens will identify mycobacterial genes involved in long-term persistence and defense against host immune mechanisms, respectively. The role of these genes in latent persistence will be assessed in the "Cornell model" of chemotherapy-induced latency and reactivation in mice. Our studies in the mouse will serve as a springboard for linked studies in humans. Relevance to human infection will be explored by analyzing the expression of per/def genes in the lungs of mice and humans via real-time RT-PCR with molecular beacons. Bacterial correlates of protection and pathogenesis will be identified by comparison of gene expression profiles in latent v. active human lesions, respectively. These studies will elucidate the host/pathogen interactions that determine the state of infection-latency or active disease-and may point the way to novel interventions against TB.